Marker and primer combination for methylation detection of cervical cancer related genes and application of marker and primer combination

Abstract

The invention relates to the technical field of biology, in particular to a marker for methylation detection of related genes of cervical cancer, a primer probe combination and application of the marker and the primer probe combination. The marker comprises GSTM5, NOVA1, TRHDE (Transient High Density Dissolved Element) and CXCL12 (CXCL12). A group of novel methylation markers for detecting the cervical cancer is obtained through screening, a group of primer combination is designed in a targeted manner, the methylation level of the markers is high, the markers can be used for methylation detection of the cervical cancer, and the screening efficiency of the cervical cancer is further improved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to markers, primer combinations and applications thereof for detecting methylation of cervical cancer-related genes. Background Art

[0002] Cervical cancer is one of the most common gynecological cancers. According to cancer statistics in 2020, there were approximately 604,000 new cases of cervical cancer and 341,000 deaths worldwide, ranking fourth in both new incidence and mortality rates among women. It is worth noting that more than 80% of cervical cancer cases worldwide occur in developing and underdeveloped countries. Among them, there were 150,700 new cases of cervical cancer and 55,700 deaths in China in 2022.

[0003] Persistent infection with high-risk human papillomavirus (HR-HPV) is considered the main risk factor for the development of cervical cancer. When the body is infected with HPV, the immune system will respond. Most infections do not cause clinical manifestations in the host and are cleared in a short period of time, but about 10% of infections will persist. It is a slow process from HPV infection to cervical cancer, which often takes years or even decades.

[0004] In order to address this global health problem, the World Health Organization (WHO) launched a global strategy to eliminate cervical cancer in 2018, aiming to reduce the global incidence of cervical cancer to less than 4 per 100,000. Among the many means to achieve this goal, population screening is one of the important intervention measures.

[0005] At present, cervical cancer screening used in clinical practice mainly adopts cervical liquid-based cytology combined with HPV testing. However, cytology testing is easily affected by human factors and has a high risk of missed diagnosis, while the specificity of HPV testing is relatively low and there is a limitation of overtreatment. More and more studies have shown that epigenetic changes are closely related to the occurrence and development of cancer. In epigenetics, DNA methylation is a common form of modification that regulates gene activity by adding methyl groups to DNA molecules without changing their sequence. In recent years, DNA methylation detection technology has continued to mature and has gradually become an emerging diagnostic method for the early stages of cancer. Among them, some methylated genes are also considered to be potential biomarkers for cervical cancer screening, such as PAX1, SOX1, JAM3, CADM1, MAL, EPB41L3, etc., but the specificity and sensitivity of these genes still need to be further tested.

[0006] In order to further improve the screening efficiency of cervical cancer, it is of great significance to explore and develop more sensitive, effective, stable, convenient and easy-to-use biomarkers in clinical practice. However, the screening of cervical cancer methylation genes provided by the present invention and their use for early diagnosis of cervical cancer have not been reported at home and abroad. Summary of the invention

[0007] In view of the above problems, the present invention provides a group of methylation genes related to cervical cancer, including GSTM5, NOVA1, TRHDE, and CXCL12; at the same time, the present invention also provides primer probe combinations for detecting these genes and uses thereof.

[0008] A first aspect of the present invention provides a marker for detecting methylation of cervical cancer-related genes, wherein the marker includes GSTM5, NOVA1, TRHDE and CXCL12.

[0009] The second aspect of the present invention provides the use of a reagent for detecting the methylation level of the above-mentioned marker in the preparation of a detection system for detecting, diagnosing, classifying or predicting, monitoring treatment, prognosing or other evaluation of cervical cancer, wherein the detection system comprises: a detection reagent, a kit or a detection device.

[0010] The third aspect of the present invention provides a primer combination, comprising A first primer set for the GSTM5 gene, which consists of a forward primer with a nucleotide sequence such as SEQ ID NO.5 and a reverse primer with a nucleotide sequence such as SEQ ID NO.6; a second primer set for the NOVA1 gene, which comprises a forward primer having a nucleotide sequence such as SEQ ID NO.7 and a reverse primer having a nucleotide sequence such as SEQ ID NO.8; a third primer set for the TRHDE gene, which consists of a forward primer with a nucleotide sequence such as SEQ ID NO.9 and a reverse primer with a nucleotide sequence such as SEQ ID NO.10; The fourth primer set for the CXCL12 gene comprises a forward primer having a nucleotide sequence such as SEQ ID NO.11 and a reverse primer having a nucleotide sequence such as SEQ ID NO.12.

[0011] The fourth aspect of the present invention provides a kit for detecting cervical cancer, comprising the above-mentioned primer combination.

[0012] In some embodiments, the kit further comprises at least one of a nucleic acid extraction reagent and a nucleic acid conversion reagent.

[0013] The fifth aspect of the present invention provides a method for detecting the methylation level of the above-mentioned marker, comprising the following steps: PCR amplification reaction; the PCR amplification uses the above-mentioned primer combination, or a kit containing the above-mentioned primer combination; SAP response; T-cut / RNase A digestion reaction.

[0014] In some embodiments, in the PCR amplification reaction, the primer concentration is 200 nM, and the reaction program is: 94°C 4 min, 94°C 20 sec, 56°C 30 sec, 72°C 1 min, 72°C 3 min, 45 cycles.

[0015] In some of the embodiments, the SAP reaction system is as follows: 1.70 μL of RNase-free ddH2O, 0.30 μL of SAP enzyme, and 5.00 μL of PCR product; the reaction procedure is 37°C, 20 min; 85°C, 5 min.

[0016] In some of the embodiments, the T-cut / RNase A digestion reaction system contains 0.64x 5x T7 polymerase buffer, 3.14 mM DTT concentration, 0.09 mg / mL RNase A, and the reaction conditions are incubation at 37° C. for 3 h.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The present invention obtains a group of new methylation markers for detecting cervical cancer through screening, and designs primer combinations specifically. The methylation level of the marker is high, and it can be used for methylation detection of cervical cancer, further improving the screening efficiency of cervical cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a schematic diagram of the screening process of methylation genes related to cervical cancer of the present invention; Figure 2 This is the ROC analysis curve of the GSE39001 dataset; Figure 3 This is the ROC analysis curve of the GSE9750 dataset; Figure 4 is the methylation level of the cervical cancer-related gene described in the present invention; Figure 5 This is a comparison of the methylation levels of different CpGs sites of the cervical cancer-related genes described in the present invention. DETAILED DESCRIPTION

[0019] The experimental methods in the following examples where specific conditions are not specified are generally carried out under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. The various commonly used chemical reagents used in the examples are all commercially available products.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0021] Example 1 Screening of methylation markers for detecting cervical cancer 1. Data acquisition and analysis of TCGA database Differentially methylated genes (DMGs) and methylation expression-related data were downloaded from DMNIVD (http: / / 119.3.41.228 / dnmivd / index / ), and DMGs were further screened out with P.adj < 0.05 as the condition. Methylation expression-related genes were then screened out based on the correlation coefficients Pearson P < 0.01 and Spearman P < 0.01. At the same time, through the GEPIA database (http: / / gepia.cancer-pku.cn), the thresholds were set to P.adj < 0.05 and log2|FC| > 1, and the analysis method was "LIMMA" differential analysis to obtain the differentially expressed genes (DEGs) data of cervical cancer. Finally, we determined the overlapping parts of the screened DMGs, DEGs, and methylation expression-related genes to obtain TCGA-MRDEGs (see Appendix for details). Figure 1 ).

[0022] 2. Data acquisition and analysis of GEO database We also used the GEO database (https: / / www.ncbi.nlm.nih.gov / geo / ) to screen GEO-MRDEGs, and selected two cervical cancer gene expression datasets (GSE39001 and GSE9750) and a methylation spectrum dataset (GSE46306) for analysis. P.adj < 0.05 was set, and the "limma" software package was used to analyze the cervical cancer sample data in GSE39001 and GSE9750 to obtain DEGs; the "minfi" software package was used to analyze the GSE46306 dataset to obtain DMGs. Finally, after determining the intersection of DEGs and DMGs, GEO-MRDEGs were obtained (see Appendix for details). Figure 1 ).

[0023] 3. Survival analysis The Kaplan-Meier tool (http: / / kmplot.com / analysis / index.php) was used to perform survival analysis for the 10 intersection MRDEGs obtained from the TCGA and GEO databases. P < 0.05 was considered statistically significant, and finally the MRDEGs closely related to the prognosis of cervical cancer patients were obtained (see Appendix for details). Figure 1 ).

[0024] 4. Screened genes The markers screened included genes GSTM5, NOVA1, TRHDE, and CXCL12; the numbers are shown in Table 1.

[0025] Table 1 Marker sequences

[0026] Example 2 Marker ROC Analysis ROC curve analysis was performed on the four MRDEGs screened from the GSE39001 and GSE9750 datasets to evaluate their diagnostic efficacy as markers for cervical cancer detection.

[0027] ROC curve, the full name of which is Receiver Operating Characteristic curve, is a tool used to evaluate the performance of diagnostic tests or prediction models. It can comprehensively consider the sensitivity and specificity of the test or model under all possible diagnostic thresholds.

[0028] For each diagnostic cutoff value, the corresponding sensitivity and specificity can be calculated. The ROC curve is plotted by using (1-specificity) as the horizontal axis and sensitivity as the vertical axis. Each point on the curve represents a specific diagnostic cutoff value, and these points are connected by line segments to form the ROC curve.

[0029] For continuous variables, the diagnostic cutoff can be arbitrarily selected within a continuous range. For ordered categorical variables, different diagnostic results can be used as diagnostic cutoffs, and each cutoff corresponds to a different sensitivity and specificity. These points are marked in a rectangular coordinate system, and adjacent points are connected with line segments to form an ROC curve for ordered categorical data.

[0030] AUC, or the area under the ROC curve, is an important indicator for measuring the performance of a diagnostic test or a predictive model. It represents the average performance of the model under all possible combinations of sensitivity and specificity. Typically, the value of the area under the ROC curve is between 0.5 and 1. The diagonal line below the curve is called the chance line, which represents the result of random guessing, that is, sensitivity equals 1-specificity. This means that the probability of being diagnosed as positive is the same for both patients and non-patients. The closer the ROC curve is to the chance line, that is, the closer the AUC value is to 0.5, the weaker the ability of the diagnostic test to distinguish between patients and non-patients. Conversely, the closer the AUC value is to 1, the higher the accuracy of the diagnostic test.

[0031] Based on the different AUC values, the diagnostic value of the diagnostic test can be evaluated: 0.50-0.70 indicates low diagnostic value; 0.70-0.90 indicates medium diagnostic value; and greater than 0.90 indicates high diagnostic value.

[0032] The ROC curve analysis results of the GSE39001 dataset are as follows Figure 2 As shown, GSTM5: AUC value was 0.9942, P value was less than 0.0001, showing extremely high diagnostic accuracy and specificity; NOVA1: The AUC value was 0.9981 and the P value was less than 0.0001, indicating that it has extremely high sensitivity in cervical cancer detection; TRHDE: AUC value was 0.9322, P value was less than 0.0001, and it had high diagnostic efficacy; CXCL12: The AUC value was 0.9690 and the P value was less than 0.0001, showing good diagnostic performance.

[0033] The ROC curve analysis results of the GSE9750 data set are as follows Figure 3 As shown, GSTM5: The AUC value was 0.9444, and the P value was less than 0.0001, which continued to show excellent diagnostic performance; NOVA1: The AUC value is 0.8157 and the P value is less than 0.0001. Although it is lower than the GSE39001 dataset, it still has a high diagnostic value. TRHDE: AUC value was 0.7955, P value was 0.0002, maintaining a high diagnostic accuracy; CXCL12: The AUC value was 0.7866 and the P value was 0.0002, which were relatively low but still had diagnostic significance.

[0034] Combined with the above analysis, the AUC values ​​of the GSE39001 dataset in this application for the four MRDEGs are all greater than 0.9, showing a high diagnostic value. This shows that these markers have high accuracy and reliability in cervical cancer detection. The AUC values ​​of the GSE9750 dataset for the four MRDEGs are also greater than 0.7. Although slightly lower than the performance in the GSE39001 dataset, they still have important application potential in cervical cancer detection. Therefore, the four screened MRDEGs showed high AUC values ​​in two independent datasets. These results support the potential of GSTM5, NOVA1, TRHDE and CXCL12 as biomarkers for cervical cancer detection. In particular, GSTM5 and NOVA1 showed extremely high AUC values ​​in both datasets, further proving the reliability of the screened genes GSTM5, NOVA1, TRHDE and CXCL12 as biomarkers.

[0035] Example 3 Verification of methylation genes related to cervical cancer According to the sequence information in Table 1, the methylation detection primer combinations of the four genes were designed, as shown in Table 2. Table 2 Primer combination sequences

[0036] 2. Agena MassArray system methylation level detection The primer combination designed in step 1 is used to detect the methylation level, including the following steps: Sample collection: Three normal cervical epithelial and three cervical cancer tissues were collected from the First Affiliated Hospital of Sun Yat-sen University for DNA methylation level detection.

[0037] (2) Sample DNA extraction and transformation 1.5 mL of cervical exfoliated cell sample was added to a sterile EP tube and centrifuged at 12000 rpm for 1.5 min, and the supernatant was removed to obtain a sample precipitate; 25 μL of proteinase K and 300 μL of lysis buffer ABL were added to the obtained precipitate in sequence, vortexed for 10 s, and incubated at 65°C for 30 min to obtain sample DNA; The extracted DNA samples were then subjected to conventional NaHSO3 treatment and briefly centrifuged at low speed for later use.

[0038] (3) PCR amplification reaction Prepare the PCR reaction solution as shown in Table 3.

[0039] Table 3 PCR reaction solution

[0040] The amplification PCR reaction program is shown in Table 4: Table 4 PCR reaction procedure

[0041] Take 3 μl of PCR product and mix with 1 μl 6*loading buffer, detect by 1.5% agarose gel electrophoresis, and observe the results after 160V and 20min.

[0042] (4) SAP reaction The SAP reaction system is shown in the following table: Table 5 SAP reaction system

[0043] Add 7 μl of SAP reaction solution to each well of the 384-well reaction plate, carefully cover with 384-well sealing film, and press each well firmly to prevent evaporation during the PCR program. After centrifugation, perform the following reaction program.

[0044] The SAP reaction procedure was 37 °C for 20 min, 85 °C for 5 min, and storage at 4 °C.

[0045] (5) T-cut / RNase A digestion reaction The T-cut / RNase A digestion reaction system is shown in Table 6: Table 6 T-cut / RNase A digestion reaction system

[0046] T-cut / RNase A digestion reaction conditions: incubate at 37°C for 3 h.

[0047] Resin purification includes the following steps, Fill the 384 / 6 MG Dimple plate with resin evenly and leave it for 10 minutes to dry; Add 16 μl of water to each well of the 384-sample plate; Place the 384 sample plate on the Dimple plate, flip it over and tap it gently to make the resin fall into each well of the sample plate; The 384 sample plate was sealed with sealing film and placed in a flip-plate centrifuge for rotation mixing at room temperature for 30 minutes.

[0048] (7) Chip spotting Start the MassARRAY Nanodispenser RS1000 to transfer the resin-purified product to the 384-well SpectroCHIP® bioarray.

[0049] (8) Massarray analysis and data output.

[0050] The spotted SpectroCHIP chip was analyzed using MALDI-TOF. The detection results were obtained using EpiTYPER™ software to obtain raw data and dot plots, and to check the integrity and accuracy of the data files.

[0051] 3. Methylation level test results of cervical cancer-related genes The methylation levels of all CPG sites in each tissue sample were statistically analyzed, and it was found that these four MRDEGs were all highly methylated in cervical cancer tissues. Figure 4 , where green is normal cervical epithelial samples and yellow is cervical cancer samples. For details on the methylation levels of different CpGs sites, see Appendix Figure 5 , where the blue one is a normal cervical epithelial sample and the yellow one is a cervical cancer sample. This shows that the marker gene loci of the present invention are suitable for methylation detection of cervical cancer.

[0052] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached claims.

Claims

1. A marker for detecting methylation of cervical cancer-related genes, characterized in that: The markers include GSTM5, NOVA1, TRHDE and CXCL12.

2. Use of the reagent for detecting the methylation level of the marker as claimed in claim 1 in the preparation of a detection system for detecting, diagnosing, classifying or predicting, monitoring treatment, prognosing or otherwise evaluating cervical cancer, the detection system comprising: A detection reagent, a kit or a detection device.

3. A primer combination, characterized in that: include A first primer set for the GSTM5 gene, which consists of a forward primer with a nucleotide sequence such as SEQ ID NO.5 and a reverse primer with a nucleotide sequence such as SEQ ID NO.6; A second primer set for the NOVA1 gene, which consists of a forward primer with a nucleotide sequence such as SEQ ID NO.7 and a reverse primer with a nucleotide sequence such as SEQ ID NO.8; a third primer set for the TRHDE gene, which consists of a forward primer with a nucleotide sequence such as SEQ ID NO.9 and a reverse primer with a nucleotide sequence such as SEQ ID NO.10; The fourth primer set for the CXCL12 gene comprises a forward primer having a nucleotide sequence such as SEQ ID NO.11 and a reverse primer having a nucleotide sequence such as SEQ ID NO.

12.

4. A kit for detecting cervical cancer, characterized in that: Comprising the primer combination as claimed in claim 3.

5. The kit according to claim 4, characterized in that The kit further comprises at least one of a nucleic acid extraction reagent and a nucleic acid conversion reagent.

6. A method for detecting the methylation level of the marker according to claim 1, characterized in that: The steps include: PCR amplification reaction; the PCR amplification uses the primer combination as described in claim 3, or the kit as described in any one of claims 4-5; SAP response; T-cut / RNase A digestion reaction.

7. The method according to claim 6, characterized in that In the PCR extension reaction, the primer concentration was 200 nM, and the reaction program was: 94°C 4 min, 94°C 20 sec, 56°C 30 sec, 72°C 1 min, 72°C 3 min, and 45 cycles.

8. The method according to claim 6, characterized in that The SAP reaction system is as follows: RNase-free ddH2O1.70 μL, SAP enzyme 0.30 μL, PCR product 5.00 μL; the reaction program is 37°C, 20 min; 85°C, 5 min.

9. The method according to claim 6, characterized in that The T-cut / RNase A digestion reaction system contained 0.64x 5x T7 polymerase buffer, 3.14 mM DTT concentration, 0.09 mg / mL RNase A, and incubated at 37° C. for 3 h.

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